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Abstract

Background

The X-linked macrosatellite DXZ4 is a large homogenous tandem repeat that in females
adopts an alternative chromatin organization on the primate X chromosome in response
to X-chromosome inactivation. It is packaged into heterochromatin on the active X
chromosome but into euchromatin and bound by the epigenetic organizer protein CTCF
on the inactive X chromosome. Because its DNA sequence diverges rapidly beyond the
New World monkeys, the existence of DXZ4 outside the primate lineage is unknown.

Results

Here we extend our comparative genome analysis and report the identification and characterization
of the mouse homolog of the macrosatellite. Furthermore, we provide evidence of DXZ4
in a conserved location downstream of the PLS3 gene in a diverse group of mammals, and reveal that DNA sequence conservation is restricted
to the CTCF binding motif, supporting a central role for this protein at this locus.
However, many features that characterize primate DXZ4 differ in mouse, including the
overall size of the array, the mode of transcription, the chromatin organization and
conservation between adjacent repeat units of DNA sequence and length. Ctcf binds
Dxz4 but is not exclusive to the inactive X chromosome, as evidenced by association
in some males and equal binding to both X chromosomes in trophoblast stem cells.

Conclusions

Characterization of Dxz4 reveals substantial differences in the organization of DNA
sequence, chromatin packaging, and the mode of transcription, so the potential roles
performed by this sequence in mouse have probably diverged from those on the primate
X chromosome.

Background

Over two-thirds of the human genome is likely to be composed of repetitive DNA [1], of which a significant proportion is tandem repeat DNA [2]. The tandem repeats consist of homologous DNA sequences arranged head to tail, and
the number of repeat units is invariably polymorphic from one individual to the next
[3]. The size of the individual repeat unit varies substantially, from the simple microsatellite
composed of individual repeat units of 1 to 6 bp spanning tens to hundreds of base
pairs [4] to those consisting of individual repeat units of several kilobases that can cover
hundreds to thousands of kilobases [5]. For some tandem repeat DNA, deciphering of function is assisted by location, such
as the alpha satellite DNA that defines active centromeres [6] to the telomeric minisatellite [7], but the roles of others in our genome remain unknown, resulting in opinions in the
past that they serve no purpose [8,9].

Despite a lack of functional understanding for these sequences, their contribution
to disease susceptibility is obvious, as is demonstrated by the devastating impact
of simple repeat expansions [10] or macrosatellite contraction [11,12].

Macrosatellites are tandem repeat DNA with some of the largest individual repeat units
(most >2 kb), which can extend over hundreds to thousands of kilobases [5,11,13-17]. Most occupy specific locations on one or two chromosomes, like the X-linked macrosatellite
DXZ4, which is unique to Xq23 [14]. Because of its physical location on the X chromosome, DXZ4 is exposed to the process
of X-chromosome inactivation (XCI). XCI is the mammalian form of dosage compensation,
an epigenetic process that serves to balance the levels of X-linked gene expression
in the two sexes [18]. It occurs early in female development and shuts down gene expression from the X
chromosome (Xi) chosen to become inactive by repackaging the DNA into facultative
heterochromatin [19]. One characteristic difference between Xi chromatin and that of the active X chromosome
(Xa) is hypermethylation of cytosine residues at CpG islands (CGIs) [20,21], but DXZ4, which is itself one of the largest CGIs in the human genome, does not
conform. Instead, DXZ4 CpG residues are hypomethylated on the Xi and hypermethylated
on the Xa [14,22]. Consistent with the DNA methylation profile of DXZ4, its nucleosomes are characterized
by the heterochromatin-associated modification histone H3 trimethylated at lysine
9 [23,24] on the Xa and the euchromatin-associated modification histone H3 dimethylated at
lysine 4 (H3K4me2) [23] on the Xi [22,25]. Furthermore, the multifunctional zinc-finger protein CCCTC-binding factor (CTCF)
[26] associates specifically with the euchromatic form of DXZ4 on the Xi [22,27]. The role DXZ4 performs on the Xi when packaged as CTCF-bound euchromatin flanked
by heterochromatin or on the Xa and male X chromosome when packaged into heterochromatin
flanked by euchromatin remains unclear. However, we have recently shown that, in humans,
DXZ4 mediates Xi-specific CTCF-dependent long-range intrachromosomal interactions
with other tandem repeat DNA [28], suggesting a structural role for DXZ4 that may orchestrate the alternative three-dimensional
organization of the Xi relative to the Xa [29]. To gain insight into DXZ4 function, we previously investigated DXZ4 in a variety
of representative primates and found that CTCF binding at the Xi was conserved, as
were the chromatin organization, expression, and arrangement of the macrosatellite
into large homogenous tandem arrays [30], but beyond the New World monkey branch, primary DNA sequence composition and tandem-repeat
unit size diverged rapidly from that observed in humans, with the notable exception
of a relatively small proportion of DXZ4 that encompassed the CTCF binding site and
promoter element [22,30]. To further our understanding of DXZ4, we extended our analysis beyond the primate
lineage in an attempt to identify a homolog of DXZ4 in mouse. Mouse has been the logical
model organism of choice for investigation of XCI, and much of what we understand
about the process has been obtained through mouse manipulations in vivo and in vitro [31]. Despite differences in the early stages of XCI between humans and mice [32], and differences in the extent of escape from XCI [33,34], identification of a mouse homolog of DXZ4 would provide a tractable system in which
to investigate function. Here we report the identification and characterization of
the mouse homolog of DXZ4. We show that DNA sequence conservation is restricted to
a short DNA sequence corresponding to the CTCF binding site, but many features of
DXZ4 differ substantially in the mouse, and as a result manipulation of mouse Dxz4
is unlikely to provide insight into all aspects of DXZ4 function in primates.

Results and discussion

Genomic organization of a mouse candidate for Dxz4

A comparison of a human DXZ4 3.0-kb tandem repeat monomer against the assembled mouse
genome (mm9) with Blast-Like Alignment Tool (BLAT) produced no significant matches
on the Ensembl genome browser [35] and a limited number of autosomal and X-linked matches on the UCSC Genome Browser
[36] (data not shown). We therefore explored conserved gene order in human and mouse to
identify a DXZ4 homolog [37]. DXZ4 resides at Xq23 [14] and is located between the t-Plastin gene (PLS3) and the Angiotensin II receptor, type-2 gene (AGTR2) (Figure 1a). Comparative analysis of human genes in the vicinity of DXZ4 in the mouse genome
revealed several differences in the gene order (Figure 1a), including a break point between the mouse PLS3 and AGTR2 orthologs Pls3 and Agtr2 and between Pls3 and the mouse ortholog of HTR2C. In mouse, the nearest proximal gene to Pls3 is Rab39b (>200 kb proximal), whereas the nearest distal gene is Tbl1x, located 1.8 Mb distal to Pls3. In humans, the respective orthologs of these two genes are located >39 Mb distal
to and >20 Mb proximal to PLS3, indicating that Pls3 alone defines the block of synteny for this region with the human X chromosome. In
primates, DXZ4 is a homogenous tandem repeat [17,30]; we therefore performed pair-wise alignments of the genomic DNA sequence upstream
of Agtr2 and downstream of Pls3 to look for evidence of tandem repeat DNA. Approximately 150 kb upstream of Agtr2, we identified an inverted repeat (Figure 1b) but no obvious tandem repeats. In contrast, pairwise alignments of the genomic DNA
sequence distal to Pls3 identified an extensive tandem repeat spanning approximately 35 kb located 19 kb 3'
to Pls3 (Figure 1c). In addition, an extensive minisatellite sequence spanning approximately 30 kb was
located a further 24 kb downstream of the tandem repeat. The minisatellite was composed
primarily of a novel gamma satellite sequence that is interrupted by several L1 and
SYNREP repetitive elements, and the sequence itself displayed an inversion almost
midway through the locus (Additional file 1). The repeat showed significant sequence matches only to the mouse X chromosome,
and no homologous repeat exists on the human or rat X chromosomes (data not shown).
We therefore focused primarily on the tandem repeat.

Figure 1.Genomic characterization of the mouse Dxz4 locus. (a) Ideograms of the human (HSAX) and mouse (MMUX) X chromosomes. Regions relevant to
the search for Dxz4 are expanded to the right of the chromosome. Genes are represented
by solid arrows pointing in the direction of transcription. Length represents extent
of the gene. Human DXZ4 is represented as the red box. The location of the putative
Dxz4 homolog and the downstream tandem repeat are highlighted proximal to mouse Pls3 as red and blue boxes, respectively. (b) Pair-wise alignment of approximately 360 kb (scale in kilobases given on the y-axis)
downstream of the mouse Agtr2 gene (20.7 to 21.1 Mb, mm9, indicated for the x-axis). Sequence similarity is shown
in blue with inverted similarity in yellow. Black bars on the top and left edges indicate
extensive repeats. (c) Pair-wise alignment of approximately 240 kb encompassing the Pls3 gene (72.9 to 73.1 Mb, mm9) and distal sequence. (d) Pairwise alignment of the 36-kb mouse Dxz4 array. The block arrows on the top and
left edges represent Dxz4 tandem repeat monomers. (e) Pairwise alignment of the largest and smallest Dxz4 monomers (block arrows on top
and left edges) highlighting the existence of an internal variable number tandem repeat
(VNTR) represented by the black arrows above the blue boxes. Perpendicular black lines
within the monomers indicate the locations of simple repeats. (f) Extended DNA fiber fluorescence in situ hybridization (FISH) of the Dxz4 array. At the top is a schematic of a single Dxz4
monomer. The regions of Dxz4 used to generate direct-labeled FISH probes are indicated
to the left (red) and right (green) of the VNTR (blue). Immediately below are examples
of hybridization results. All pairwise alignments used the DNA sequence compared with
a repeat-masked version of itself with the exception of that shown in (c), which compared
non-repeat-masked sequences to show the inverted satellite repeat. Alignments were
all made with YASS [71], and the output was pseudocolored to avoid red-green.

Additional file 1.Genomic organization and expression of the downstream tandem repeat. The pair-wise alignment and repeat content of the Ds-TR as well as expression as
demonstrated by RT-PCR.

We next checked to see how frequently a tandem repeat of comparable size (35 kb) occurred
on the mouse X chromosome to see if detection of such a sequence downstream of Pls3 would likely occur by chance. Pair-wise alignments along the length of the mouse X
chromosome indicated that large tandem repeats are not common (Additional file 2), supporting the possibility that this might be the mouse homolog of DXZ4.

Pair-wise alignment of the tandem repeat sequence revealed that, unlike DXZ4 in primates,
where repeat units are very similar in size within a species [17,30], the individual repeating units of the mouse tandem repeat varied from 3.8 to 5.7
kb (Figure 1d). Closer examination showed that the size variation was accounted for by the presence
of an internal variable number tandem repeat (VNTR) of an approximately 900-bp sequence
present as between one and three copies per monomer (Figure 1e). As in primate DXZ4 [14,17,30], less than 6% of the smallest monomer DNA sequence (3.8 kb) was repeat masked, and
all of the masked regions corresponded to simple repeats. Examination of the largest
monomer (5.7 kb) revealed that the first 147 bp of the internal VNTR was derived from
an ERV class II long terminal repeat and that the other edge of the VNTR is defined
by a simple repeat. The location of these repeat sequences may contribute to the observed
copy-number variation. Three other defining features of human DXZ4 were examined for
the novel mouse tandem repeat: CpG content, sequence variation between monomers, and
size of the tandem array. Human DXZ4 DNA is 62.2% GC, contains 186 CpG dinucleotides
per monomer [38], and shows less than 1% sequence divergence between adjacent monomers [17]. In contrast, the mouse 3.8-kb monomer is 53.4% GC, contains 36 CpG dinucleotides,
and shows greater than 5% sequence divergence from other monomers in the tandem array.
In primates, DXZ4 is composed of as many as 100 repeat units spanning hundreds of
kilobases on the X chromosome [14,17]. In the current build of the mouse genome, the tandem repeat is composed of approximately
seven repeat units. Given the inherent difficulty with the computer-based assembly
of tandem repeats [39], the actual array could be more extensive. We have previously used extended DNA fiber
fluorescence in situ hybridization (FISH) to confirm tandem arrangement and copy-number variation of human
DXZ4 [17]. We applied the same procedure to examine such variation in the mouse tandem repeat,
revealing approximately six tandem repeats in two independent mouse cell lines (Figure
1f). This result suggested that the mouse tandem repeat is relatively small, and the
presence of the tandem repeat and extensive flanking DNA sequences entirely within
the inserts of at least ten independent mouse bacterial artificial chromosomes from
three different libraries derived from two Mus musculus subspecies lends additional support (Additional file 3). The logical interpretation of these observations was that the mouse sequence downstream
of Pls3 is a tandem repeat but that the overall copy number of repeat units is low, resulting
in a smaller array than in primates. Despite these differences from primate DXZ4,
the tandem repeat remains a good candidate for the mouse homolog and from this point
forward is referred to as Dxz4.

Expression of Dxz4

Primate DXZ4 is expressed, and all regions of a monomer can be detected in complementary
DNA (cDNA) [17,22,30]. Six regions of Dxz4 were assessed in cDNA from several different mouse total-RNA
sources. The example shown in Figure 2a indicates that mouse Dxz4 was also expressed and that all parts of the Dxz4 monomer
are transcribed into RNA. This result was confirmed by RNA FISH showing readily detectable
Dxz4 primary transcript by means of direct-labeled Dxz4 probes (Figure 2b). In humans, DXZ4 is primarily transcribed from one strand (since designated the
sense strand), but antisense transcript can be detected in females and is therefore
interpreted as originating from the Xi [22]. Our previous data showed that only sense transcript could be detected in macaque
[30]. To assess the relative frequencies of sense and antisense transcription of Dxz4,
we primed male and female cDNA from total RNA using oligonucleotides that would prime
sense or antisense cDNA synthesis. As in macaque, only sense transcript was readily
detected (Figure 2c). In humans, DXZ4 transcript can be detected from the Xa and the Xi [17,22], although expression in macaque is almost exclusively restricted to the Xa [30]. RNA FISH was performed on female mouse cells with a direct-labeled Dxz4 probe and
a probe to the X inactive specific transcript (Xist) [40,41] to define the location of the Xi (Figure 2d). As in macaque, Dxz4 could only be readily detected from the Xa (Figure 2e). Collectively, our interpretation of these data is that expression of Dxz4 is restricted
to the Xa allele and from one strand only.

Figure 2.Characterization of unspliced Dxz4 transcript. (a) Schematic map of a Dxz4 monomer. The internal VNTR is represented by the black box.
Below it are indicated six intervals (i to vi) assessed by reverse-transcription PCR
(RT-PCR). The RT-PCR results for i to vi are given as images of ethidium bromide-stained
agarose gels for NIH/3T3 complementary DNA (cDNA). Samples include water (W), RNA
incubated with (+RT) and without (-RT) reverse transcriptase, and genomic DNA. (b) RNA FISH results of direct-labeled Spectrum-Orange or Spectrum-Green probes for regions
i to vi in NIH/3T3 cells. Signals are indicated by white arrows merged with DAPI (black
and white). (c) Strand-specific quantitative RT-PCR analysis of Dxz4 expression in two independent
male and female samples. Graph shows fold expression of dxz4 in sense (left) and anti-sense
(right) primed cDNA relative to cDNA prepared with no gene-specific primer. Error
bars show standard deviation. (d) RNA FISH analysis of unspliced Dxz4 (red) and Xist RNA (green) merged with DAPI (black
and white) in female cells. Dxz4 indicated by the white arrowheads and inactive X
chromosome-specific transcript (Xist) by the white arrows. (e) Frequency of Dxz4 RNA FISH signals overlapping Xist in female cells.

Examination of the GenBank mouse mRNA annotation for the Dxz4 locus on the UCSC Genome
Browser [36] revealed the presence of two alternatively spliced transcripts spanning Dxz4. Both
transcripts originate at an exon almost 2.2 kb from the distal edge of the array (Figure
3a). The transcript then spliced to the same 163-nucleotide sequence within each of
the monomers before splicing to an exon located 1.1 kb proximal to the array. One
of the two spliced transcripts proceeded to be spliced to two additional exons approximately
16.0 kb downstream, whereas the other read through the splice site before terminating
after a further 2.0 kb. To confirm the existence of the spliced forms of Dxz4, we
performed reverse-transcription PCR (RT-PCR) between different combinations of the
exons. The anticipated product was detected for each of the RT-PCR experiments (Figure
3b). Furthermore, the RT-PCR confirmed that the transcript contains multiple copies
of the 163-nucleotide exon as can be seen from the laddered effect of progressively
larger PCR products (see the PCR of exon 1 to 2 or 2 to 3 as examples). Furthermore,
this exon was also alternatively spliced with some transcripts omitting one or more
163-nucleotide exons. This result could be observed as smaller laddered bands when
RT-PCR was performed across the entire array (Figure 3b, exon 1 to 9/10).

Figure 3.Expression of spliced Dxz4 and promoter characterization. (a) Schematic map of the Dxz4 region representing 72.95 to 73.01 Mb of the mouse X chromosome
(mm9). The map is inverted for simplicity and the distal direction toward Pls3 indicated. Open block arrows represent Dxz4 monomers. A downstream CGI is indicated.
Immediately below is a map indicating location and type of repeat elements for the
interval: LINE, long interspersed nuclear element; LTR, long terminal repeat; SINE,
short interspersed nuclear element. Below that are the maps of two putative alternatively
spliced transcripts based on expressed sequence tag evidence. (b) Confirmation of spliced transcripts by RT-PCR. Each of the seven panels is an image
of an ethidium bromide-stained agarose gel showing RT-PCR results for PCR between
the exons indicated above. To the left of each image is the predicted product size.
Samples include water control (W) and RNA incubated with (+RT) and without (-RT) reverse
transcriptase. (c) DNA sequence feature map of the 1.3-kb region immediately upstream of Dxz4 exon 1
(green). Repetitive elements are indicated above the corresponding colored boxes.
Immediately below are the regions cloned upstream of a promoterless luciferase reporter
gene: construct A (Con.A) and construct B (Con.B). (d) Luciferase activity measured in NIH/3T3 cell extracts 72 hours after transfection
with the promoterless luciferase vector (pGL4.10) or the same vector containing inserts
for construct A or B. Fold activation of luciferase is shown to the left. Data represent
the mean and standard deviation of replicate experiments each performed in triplicate.

Both the spliced and unspliced transcripts corresponded to the sense transcript, and
therefore probably originated from a common promoter, unlike human DXZ4, which contains
a region with promoter activity within each monomer [22]. Examination of histone modification profiles from the Encyclopedia of DNA Elements
(ENCODE) [42] revealed a distinct peak of histone H3 trimethylated at lysine 4 (H3K4me3) [43] in the vicinity of exon 1 (data not shown). H3K4me3 is a modification associated
with transcriptional start sites [44]. We therefore cloned the DNA sequence 5' of Dxz4 exon 1 immediately upstream of a
promoterless luciferase reporter gene. Two constructs were generated. The first consisted
of a 1.2-kb sequence that contained several repetitive elements that are located immediately
upstream of exon 1 (Figure 3c). The second construct consisted of a 238-bp unique sequence 5' of exon 1. Robust
promoter activity was detected for both constructs (Figure 3d); the highest activity consistently originated from the smaller unique sequence construct,
confirming the location of the minimal Dxz4 promoter.

We next checked to see if the Dxz4 tandem repeat possessed intrinsic promoter activity
like human DXZ4 [22]. Two overlapping fragments encompassing a complete Dxz4 monomer were PCR amplified
(Additional file 4), TA cloned and sequence verified. The DNA was then subcloned upstream of the promoterless
luciferase reporter gene and assessed for promoter activity alongside the Dxz4 minimal
promoter described above. Neither Dxz4 fragment showed obvious activity compared to
the Dxz4 minimal promoter that consistently activated luciferase greater than 200-fold
over the empty vector (Additional file 4). Therefore, our interpretation of this result is that both the spliced and unspliced
Dxz4 transcripts likely originate from transcription initiating from the minimal promoter.
Consequently, it should be possible to detect by RT-PCR a transcript that spans exon
1 directly to the tandem repeat (Additional file 5). Despite the relatively large size (approximately 2.5 kb) and proximity to the very
5' end of the message, this transcript can be detected in cDNA (Additional file 5).

When the H3K4me3 profile of Dxz4 was examined, an additional major peak was noticed
immediately distal to the downstream inverted tandem repeat (Ds-TR; data not shown),
suggesting promoter activity within this region and the possibility that, like Dxz4,
the Ds-TR is expressed. RT-PCR confirmed expression of Ds-TR in both male and female
samples (Additional file 1).

CpG methylation analysis in and around Dxz4

DXZ4 is unusual in that CpG dinucleotides are hypermethylated on the Xa but hypomethylated
on the Xi [14,22], a trait that is conserved in macaque [30]. We selected several regions in and around Dxz4 at which to determine and compare
the CpG methylation profiles of males and females (Figure 4a). These sites included the Dxz4 promoter, a region of relatively high CpG incidence
within the Dxz4 internal VNTR, a CGI immediately downstream of the Dxz4 array (DD-CGI),
and two regions in the vicinity of the H3K4me3 peak adjacent to the Ds-TR.

Figure 4.DNA methylation of elements in the vicinity of Dxz4. (a) Schematic map of the region encompassing Dxz4 and the downstream satellite repeat
(diverging open arrows). Left-pointing arrows represent Dxz4, and the location of
the Dxz4 promoter and CGI are indicated. The red boxes indicate regions assessed for
DNA methylation by PCR of bisulfite-modified DNA, cloning, and sequencing. The location
of bisulfite analysis within the Dxz4 array is shown for a single monomer immediately
below the array. (b) Cytosine methylation at CpG dinucleotides for the five regions shown in (a). Data
are given for two independent males (top) and two independent females (bottom). Methylated
cytosine is represented by a black circle whereas unmethylated is represented by an
open circle. DNA variants that result in a sequence that is no longer a CpG are represented
by dashes. Each row of circles represents DNA sequence obtained from a single clone,
and each set of data consists of at least nine independent clones.

The Dxz4 promoter (Figure 4b, far right) showed a significantly higher percentage of CpG methylation in females
than in males (P = 0.0052, two-sample t-test). This result is consistent with our expression analysis (Figure 2), suggesting that transcription of Dxz4 is subject to XCI [20,21] and explaining why Dxz4 transcript was only detected from the Xa (Figure 2e).

Males and females did not differ significantly in methylation of the sequence closest
to the Ds-TR (profile on far left in Figure 4b; P = 0.7580) or in the region immediately distal to it (P = 0.0577), but the two regions differed vastly; the proximal sequence was almost entirely
methylated, and the distal sequence hypomethylated, on both X chromosomes. Both sites
overlap a broad signal of H3K4me3 (data not shown), but examination of other ENCODE
features [42] at these two regions revealed that the hypomethylated sequence overlapped a major
peak of occupancy for Ctcf [45] and a DNaseI hypersensitive site [46], whereas the hypermethylated site did not (Additional file 6). Binding of Ctcf to target sites containing CpG is sensitive to methylation [47,48]. The hypomethylation in males and females suggests that Ctcf has the potential to
bind this region on both the Xa and the Xi.

Additional file 6.CpG methylation relative to Ctcf and DNaseI hypersensitivity. The location of a Ctcf and DNaseI peak relative to CpG methylation immediately adjacent
to the Ds-TR.

Males and females did differ significantly in CpG methylation at the Dxz4 array (P = 0.0027) similar to what we and others have reported for primate DXZ4 [14,22,30]. However, many sites of CpG residues predicted on the basis of the reference genome
sequence (mm9) are not conserved, as demonstrated by the numerous gaps in the bisulfite
profiles. Methylated cytosine in CpG is prone to mutation by deamination, whereas
mutation rates of unmethylated CpG are lower [49]. As a consequence, hypomethylated CGIs are evolutionarily conserved [50]. The apparent lack of conservation of CpG dinucleotides at Dxz4 is consistent with
the overall hypermethylated profiles (Figure 4b). This situation differs from that of primate DXZ4, where CpG residues are highly
conserved [22,30], consistent with evolutionary maintenance of DXZ4 as an extensive CGI [50].

Furthermore, males and females did differ significantly in methylation at DD-CGI (P = <0.0001); more hypomethylated clones were obtained from the female samples (Figure
4b). Our interpretation of these data is that DD-CGI is hypomethylated on the Xi. DD-CGI
spans 333 bp and contains 40 CpGs on the basis of the C57BL/6J reference genome sequence
(mm9). None of the genomic feature annotations generated by ENCODE [42], including Ctcf, highlight DD-CGI, and therefore the significance of Xi hypomethylation
remains unclear.

Histone methylation and Ctcf association with sequences in the vicinity of Dxz4

Next we sought to complement the DNA methylation analysis by examining histone methylation
and Ctcf binding in and around Dxz4. Several sites were selected, including the Dxz4
promoter, the Dxz4 VNTR region, DD-CGI, Ds-TR, and the Pls3 promoter as a control for mouse genes subject to XCI [34] (Figure 5a).

Figure 5.Characterization of chromatin in the vicinity of Dxz4. (a) Schematic map of the region encompassing Dxz4 and the downstream satellite repeat
(diverging open arrows). Left-pointing facing arrows represent Dxz4, and the location
of the CGI and promoters for Dxz4 and Pls3 are indicated. The angled double strike through the map between the Dxz4 and Pls3 promoters represents an approximately 114-kb gap. The red boxes indicate the regions
assessed by chromatin immunoprecipitation (ChIP)-PCR. (b) Graphs showing results of ChIP assayed by quantitative PCR. The mean and standard
deviation for the ChIP elution (IP) and for a negative control rabbit serum (RS) are
shown as percentage of the input. For H3K4me2 and H3K27me3 at the Dxz4 promoter (Dxz4-Prom)
and Pls3 promoter (Pls3-Prom), data for one male and one female are shown. For H3K4me2 and
Ctcf at Dxz4, DS-TR and DD-CGI, data are shown for two independent male and female
samples. (c) Pie charts showing the percentage of C57BL/6J (B6) or castaneous (Cast) informative
allele calls for Ctcf ChIP-Seq fragments for Dxz4 and the downstream tandem repeat
(Ds-TR) Ctcf binding sites.

Consistent with the expression analysis (Figure 2) and CpG methylation (Figure 4b), the Dxz4 promoter was characterized by the euchromatin mark H3K4me2 in male and
female cells, whereas the facultative heterochromatin marker histone H3 trimethylated
at lysine 27 (H3K27me3) was only a feature of the female samples (Figure 5b). The same profile is obtained for the Pls3 promoter, which is subject to XCI in mouse [34]. Given that genes on the Xi are silenced by H3K27me3 [51,52], these data further support the conclusion that Dxz4 expression is subject to XCI.

In primates, H3K4me2 is a feature of DXZ4 on the Xi [22,30], although this modification can be detected on the male X at low levels in some individuals
and as a result of cellular transformation [53]. In contrast, H3K4me2 was readily detected at Dxz4 in males and females (Figure 5b), another difference between mouse and primate DXZ4. Somewhat surprisingly, given
the methylation profile at DD-CGI (Figure 4b), H3K4me2 could also be detected at this site in males and females. One possible
explanation is that the DD-CGI is located within the transcriptional unit of one of
the spliced Dxz4 transcripts (Figure 3a). Therefore, the detection of the euchromatin mark may reflect variable levels of
H3K4me2 in the body of active genes [44].

A defining feature of primate DXZ4 is the association of CTCF with the Xi allele [22,30]. Ctcf was readily detected at Dxz4 in multiple independent female samples, but Ctcf
was also detected, albeit at lower levels, in some but not all males (Figure 5b and data not shown). To investigate further the relationship between Ctcf and Dxz4
on the Xa and Xi, we examined DNA sequence reads from Ctcf chromatin immunoprecipitation
(ChIP) combined with next generation sequencing (ChIP-Seq) performed on trophoblast
stem cells (TSCs), which are derived from the extraembryonic material and undergo
imprinted XCI with preferential inactivation of the paternal X chromosome [54]. The TSCs were derived from a cross of a male C57BL/6J (BL6) with a female castaneous
(cast) mouse. As a result, the BL6 X chromosome will be the Xi. ChIP-Seq reads were
compared to BL6 and cast variant sequences for the Dxz4 interval assessed by ChIP-PCR
and, where informative, were designated as originating from the Xa (cast) or Xi (BL6).
Of 152 ChIP-Seq reads, almost half were assigned to the Xa and half to the Xi (Figure
5c), consistent with detection of Ctcf at the Xa in some males. One interpretation of
these data is that Ctcf binds Dxz4 at the Xa and Xi equally, but not detecting Ctcf
at Dxz4 in all males even when it is readily detected in the same samples at a known
Ctcf binding site within the H19 imprinted control region [47,48] suggests that binding of Ctcf to Dxz4 varies. This result could reflect subtle differences
in CpG methylation (compare the two male bisulfite profiles in Figure 4b), strain or cell-type differences. Nevertheless, these observations are consistent
with the differences we report above for Dxz4 chromatin organization at the Xa and
Xi between mouse and primates. Notably, the association of Ctcf within the VNTR region
means that although the array itself is relatively small, the potential Ctcf occupancy
is higher than one per repeat monomer.

As mentioned above, the unique sequence (Ds-TR) located immediately distal to the
large inverted satellite repeat (Figure 5a; Additional file 1) is characterized by DNaseI hypersensitivity and Ctcf binding (Additional file 6). Ctcf ChIP-PCR confirmed association with this sequence in males and females (Figure
5b), and as anticipated given the CpG hypomethylation (Figure 4b), the region was characterized by H3K4me2. To determine whether Ctcf at Ds-TR is
associated with the Xa alone or with Xa and Xi, we used informative BL6 and cast SNPs
to assign Ctcf ChIP-Seq reads to their X chromosome of origin. Unlike Dxz4, Ctcf at
Ds-TR was biased toward the Xa but could also bind the Xi to a lower extent (Figure
5c).

Conservation of a large tandem repeat downstream of PLS3 in mammals

Thus far we have shown that, as in primates [14,17,22,30], a large tandem repeat is present downstream of Pls3 on the mouse X chromosome despite extensive shuffling of the locations of genes from
the same interval (Figure 1a). We sought to determine whether a tandem repeat was present downstream of PLS3 in a diverse set of mammals for which genome assemblies were sufficiently complete.
Pairwise alignment of genomic sequence distal to PLS3 was performed for seven different mammals. Each revealed the presence of a tandem
repeat within 28 to 110 kb of the 3' end of PLS3 (Figure 6).

Figure 6.Identification of a tandem repeat downstream of PLS3 in eight different mammals. Pairwise alignment of genomic DNA sequence encompassing and extending downstream
of PLS3 for each mammal (labeled above each plot). The structure and location of PLS3 is indicated on the top and left edge of each alignment. Distance in kilobases is
indicated to the right of each plot. The distance between the 3' end of PLS3 and the downstream tandem repeat is highlighted above each plot. The extent of the
tandem repeat is highlighted by the black bar above and to the left of each plot.
Arrows pointing down from the top or rightward from the left edge indicate gaps in
the genome assembly.

Conservation of the CTCF binding sequence at DXZ4

Previously we have shown that a region encompassing the CTCF binding site is conserved
in primates, but outside of this interval divergence of the sequence of DXZ4 and size
of the individual tandem repeat unit increases substantially with distance down the
primate tree [30]. Focusing only on this region, we identified 74% nucleotide identity over 100 bp
between human DXZ4 and the VNTR region within each mouse Dxz4 monomer. Similar levels
of nucleotide identity over the same interval were identified within the tandem repeat
DNAs shown in Figure 6. We used this interval to extract homologous DNA sequence entries from 25 different
mammals before aligning all of the sequences. Most of the mammals examined formed
clades corresponding to their respective orders and suborders, such as the primates,
which all branch from a single node (Figure 7a). These data support evolution of DXZ4 from a common ancestor in a manner analogous
to that of coding sequences. Close examination of the DNA sequence alignment revealed
a subregion of the conserved DNA sequence in which several nucleotides were identical
in all 25 mammals. A 34-bp sequence encompassing all invariable nucleotides was extracted
from each sequence and used to generate a position weight matrix [55] that clearly revealed the nonrandom nature of this sequence (Figure 7b). Given that this sequence is entirely contained within the region assessed by PCR
in primate CTCF ChIP [22,30], mouse Ctcf ChIP (Figure 5b), and mouse Ctcf ChIP-Seq (Figure 5c), the position weight matrix sequence was compared with a previously defined Ctcf
consensus sequence [47], and as can be seen in Figure 7b, the most conserved DXZ4 sequence in all mammals examined was a good match to this
consensus.

Figure 7.Identification of a conserved DNA sequence element with homology to a CTCF consensus
sequence in mammalian DXZ4. (a) Schematic representation of a mouse Dxz4 monomer. The green arrowhead indicates the
spliced exon. The blue vertical bars indicate repeat-masked sequence. The black bar
represents the VNTR. The yellow box within the VNTR (bases 919 to 1,061) represents
the conserved Dxz4 sequence. This sequence was used to align to the corresponding
sequences from the mammals listed to generate the cladogram. The tree image was generated
with MUSCLE version 3.8 [72] and ClustalW2 [73]. Classification of the groups is given to the right. (b) Schematic representation of a mouse Dxz4 monomer as above. The yellow box within the
VNTR (bases 978 to 1,011) represents the DNA sequence that contains nucleotides invariable
in all mammalian DXZ4 sequences assessed. This 34-bp sequence from each mammal was
used to generate the position weight matrix through WebLogo [55]. Beneath the matrix is a previously determined Ctcf consensus sequence that is adapted
from Martin et al. [47]. Note that the position weight matrix is the reverse complement of that shown in
the referenced manuscript.

The Ctcf match to the conserved sequence only accounts for bases 3 to 21, yet conservation
of DNA sequence across the diverse group of mammals extends for an additional 13 bp.
It is conceivable that this extended conservation reflects retention of an additional
binding motif(s) for other DNA binding protein(s). To explore this possibility, the
consensus sequence was compared to motifs in JASPAR [56]. Two motifs showed good matches to this region. The first is a 9 out of 10 base match
to the recently determined mouse consensus for the CCAAT/enhancer-binding protein
alpha (Cebpa) [57], whereas the second is a match (9 out of 9) for the human consensus for ETS-domain
protein 4 (ELK4) [58] (Additional file 7). Cebpa is an essential basic-leucine zipper DNA binding protein that performs essential
roles in the development of myeloid cells [59] and in liver function [60]. ELK4 is a ubiquitous serum response factor accessory protein [61] that is found at many locations in the genome [62]. Whether either protein binds to Dxz4 has yet to be determined, but given the broad
cross-species conservation of the DNA sequence and good matches with each DNA binding
consensus sequence [57,58], both are candidates worthy of further investigation.

Conclusions

Comparative genomics is a powerful means of uncovering important functional DNA elements
through DNA sequence conservation [63], but identification of mouse Dxz4 was initially discovered not through primary DNA
sequence conservation but instead through conservation of DNA sequence organization
within a syntenic region of the mouse genome. This work led to the subsequent identification
of DXZ4 in a diverse group of distantly related mammals. DNA sequence comparisons
revealed a highly conserved region within each DXZ4 monomer that corresponds to the
CTCF binding motif that is bound by CTCF in all mammals tested thus far. Furthermore,
the highly conserved sequence immediately adjacent to the Ctcf consensus site suggests
a second DNA binding protein may associate alongside Ctcf. Therefore, on the basis
of conservation, several features of DXZ4 appear to have functional importance in
eutherian mammals: CTCF binding, tandem-repeat organization, expression, and location
downstream of PLS3.

In primates CTCF association with DXZ4 is almost exclusively Xi-specific [22,30], yet the analysis of mouse Dxz4 we report here suggests that its chromosome specificity
is not as clearly defined; it apparent binds to both the Xa and the Xi to varying
degrees. Primates and mouse appear to differ in several other aspects of DXZ4. First,
primate DXZ4 is composed of a large number of tandem repeat units in which adjacent
repeat monomers share very high DNA sequence identity and length [17,30]. The same is not true of mouse Dxz4. The tandem array is small in comparison, and
individual repeat monomers display pronounced sequence variation and the presence
of an internal VNTR. Perhaps near-identical sequence composition and monomer size
are a prerequisite for expansion, such as the observed complex gene conversion mechanisms
reported for minisatellites [64] or through alternative processes such as intrachromatid recombination or unequal
exchange [65]. Second, DXZ4 DNA sequence is GC-rich in primates [14,17,22,30] but not in mouse. Third, DXZ4 in humans contains a DNA sequence with inherent promoter
activity in each monomer [22]. This sequence is not conserved in mouse and intrinsic promoter activity is not obvious
within the Dxz4 monomers. Instead a promoter located to one side of Dxz4 drives transcription
across the entire array, but tandem repeat units in several other mammals do show
substantial DNA sequence homology to human DXZ4 beyond the CTCF binding region encompassing
the promoter sequence. These include cat, dog, horse, elephant, dolphin, microbat,
rabbit, and flying fox (data not shown), suggesting that these mammals will likely
retain internal promoter activity negating the need for the external promoter. Fourth,
although all DXZ4 examined is transcribed [17,22,30], at least some mouse Dxz4 is spliced, a feature not observed in primates. Finally,
euchromatin is largely restricted to DXZ4 on the Xi in primates [22,30] yet H3K4me2 is a feature of Dxz4 on the Xa in mouse. One feature that is consistent
between the mouse and primate macrosatellite is significantly higher incidence of
CpG hypomethylation in females that we interpret as originating from the Xi. Compared
to primates, however, the overall profile is more methylated in mouse relative to
primates [14,22,30]. Conceivably, the hypermethylation of Dxz4 combined with lower overall GC content
is accelerating mutation of CpG dinucleotides [66].

Collectively, these observations suggest that the functions performed by DXZ4 in primates
are not all necessarily conserved in mouse. We hypothesize that primate DXZ4 has important
but distinct roles on the Xa and Xi that both necessitate a large homogenous tandem
array. On the Xa this role involves expression and packaging into heterochromatin.
Given the extreme copy-number variation of DXZ4 [14,17], the macrosatellite could conceivably modulate the transcription of the adjacent
PLS3 gene, which shows considerable variation in expression levels between individuals
[67]. In contrast, on the Xi a euchromatic organization bound by CTCF is required. The
fact that CTCF is central to mediating genome organization [68], and that, at least in humans, CTCF-bound DXZ4 mediates Xi-specific long-range intrachromosomal
interactions with other Xi-specific CTCF-bound tandem repeats [28] suggests that DXZ4 performs a structural role on the Xi. Mouse Dxz4 may or may not
perform either function, and the difference could contribute to some of the observed
differences between the biology of the human and mouse X chromosome, such as the variable
escape of PLS3 expression from the Xi in humans [33] but not in mouse [34]. The distinct differences between DXZ4 and Dxz4 suggest that, if Dxz4 performs a
similar function, it has evolved alternative strategies in order to do so. Nevertheless,
the evolutionarily constrained association of CTCF/Ctcf with mammalian DXZ4 appears
central even if conservation of function is not.

Bisulfite modification of DNA, cloning and sequencing

Genomic DNA was isolated from primary cells with the NucleoSpin Tissue kit (Machery-Nagel,
Bethlehem, PA, USA). Genomic DNA was isolated from mouse tail snips by standard techniques
[69]. Unmethylated cytosines were converted to uracil with the EpiTect bisulfite modification
kit (Qiagen, Valencia, CA, USA). Bisulfite-modified DNA was used as a template for
PCR with OneTaq® master mix (NEB, Ipswich, MA, USA) and the primers listed in Additional file 8. PCR products were cloned into pDrive TA vector (Qiagen), and positive clones sequenced
(Eurofins MWG Operon, Huntsville, AL, USA) and analyzed with Sequencher 5.0 (Gene
Codes Corp., Ann Arbor, MI, USA). Statistically significant differences in methylation
between males and females were determined as follows. The percent methylation for
individual clones (a single horizontal line in the profiles) was determined and the
mean and standard deviation was calculated for the males and females. These were compared
using the two-tailed t-test with differing variance as described previously for methylation profiles [70].

Additional file 8.Table listing all oligonucleotides used in this study.

RNA and extended DNA fiber FISH

Mouse Dxz4 fragments were PCR amplified and cloned into the TA vector pCR2.1 (Life
Technologies Corp.) before sequence verification. Direct-labeled FISH probes were
generated from Dxz4-pCR2.1-isolated DNA with SpectrumOrange™ or SpectrumGreen™ and
a nick translation kit (Abbott Molecular, Abbott Park, IL, USA). Probes were heat
inactivated at 68°C for 10 minutes before ethanol precipitation and resuspension in
Hybrisol VII (MP Biomedicals, Santa Ana, CA, USA). RNA FISH was performed on cells
grown directly on microscope slides. Cells were rinsed with 1× phosphate-buffered
saline (PBS) before being fixed and extracted for 10 minutes at room temperature in
3.7% formaldehyde, 0.1% Triton X-100 in 1× PBS. Slides were rinsed twice in 1× PBS
before dehydration for 3 minutes in 70% and 100% ethanol before being air-dried. Probes
were denatured in a thermal cycler at 72°C for 10 minutes before the temperature was
reduced to 37°C, at which point the probe was applied directly to the slide, sealed
under a cover glass, and hybridized overnight at 37°C. Cover slips were removed and
the samples washed twice at room temperature for 2 minutes each in 50% formamide/2
SSC, once for 3 minutes at 37°C in 50% formamide/2× SSC, and once for 3 minutes at
37°C in 2× SSC before addition of ProLong® Gold antifade reagent supplemented with DAPI (Life Technologies Corp.). Mouse extended
DNA fibers were prepared and FISH performed essentially as previously described [17]. Images were either collected with a Zeiss Axiovert 200 M fitted with an AxioCam
MRm and managed with AxioVision 4.4 software (Carl Zeiss microimaging) or collected
with a DeltaVision pDV. Delta Vision images were deconvolved with softWoRx 3.7.0 (Applied
Precision, Issaquah, WA, USA) and compiled with Adobe Photoshop CS2 (Adobe Systems).

Standard and strand-specific cDNA preparation and PCR

Total RNA was extracted from cells with the NucleoSpin RNA II kit (Machery-Nagel).
For standard RT-PCR, first-strand cDNA was prepared from 2 μg of total RNA with random
hexamers with and without M-MuLV reverse transcriptase (RT) according to the manufacturer's
instructions (NEB). cDNAs prepared with and without RT were used as templates for
PCR with either OneTaq® master mix (NEB) or HotStar Taq (Qiagen) with the primers listed in Additional file
8. PCR was performed using an initial denaturation of 10 minutes at 94°C, followed
by 35 cycles of: 94°C for 30 seconds, 58°C for 30 seconds and 72°C for 30 seconds
for all products of up to 750 bp, 1 minute for all products up to 1,250 bp and 1 minute
30 seconds for products up to 2 kb. The cycling was followed by 10 minutes at 72°C
before holding at 15°C. Strand-specific cDNA was prepared as above except that first-strand
cDNA was primed with 1.5 pmol of a specified oligonucleotide (Additional file 8) in place of random hexamers and an additional control that included RT but no oligonucleotide
that is used to determine the background levels of cDNA synthesized in the absence
of a gene-specific primer. Strand-specific cDNA was assessed by quantitative RT-PCR
using the primers given (Additional file 8) with a SYBR-Green qPCR Mastermix (SABiosciences, Qiagen) on a CFX96 (Biorad, Hercules,
CA, USA). PCR was performed using an initial 10-minute denaturing step at 95°C followed
by 40 cycles of: 15 seconds at 95°C, 30 seconds at 60°C and 30 seconds at 72°C. The
cycle was followed by a melt-curve. PCR was performed in triplicate and the transcript
level determined relative to background.

Promoter luciferase assay

DNA fragments initiating in and extending upstream of Dxz4 exon 1 were generated by
PCR with Platinum®Taq (Life Technologies Corp.; 94°C for 2 minutes followed by 40 cycles of: 94°C for
30 seconds, 58°C for 30 seconds and 68°C for 1 minute 20 seconds for construct A or
68°C for 30 seconds for construct B) and cloned into pDrive (Qiagen). Inserts were
verified by DNA sequencing before subcloning into the KpnI and XhoI sites of pGL4.10[luc2] (Promega, Madison, WI, USA). The Dxz4-promoter pGL4.10[luc2]
firefly luciferase reporter constructs were co-transfected in triplicate on two separate
occasions with the Renilla-luciferase expression vector pGL4.74[hRluc/TK] (Promega) into NIH/3T3 cells by means
of Lipofectamine 2000 (Life Technologies Corp.). Cells were assayed for luciferase
activity on a Glomax-20/20 Luminometer (Promega) 72 hours after transfection with
the dual-luciferase reporter assay system, according to the manufacturer's recommendations
(Promega).

ChIP and analysis

Standard ChIP was performed on mouse cells essentially as described previously [22] except that formaldehyde cross-linking was with 0.75% formaldehyde rather than 1.0%.
Chromatin was sheared with a Bioruptor (Diagenode, Denville, NJ, USA) set at 8 cycles
of 30 seconds on and 30 seconds off on high setting. Rabbit polyclonal antibodies
used were all obtained from Millipore (Billerica, MA, USA) and included anti-H3K4me2
(07-030), anti-H3K27me3 (07-449), and anti-CTCF (07-729). ChIP was assessed by quantitative
PCR using the primers given (Additional file 8) with a SYBR-Green qPCR Mastermix (SABiosciences, Qiagen) on a CFX96 (Biorad). PCR
was performed using an initial 10-minute denaturing step at 95°C followed by 40 cycles
of: 15 seconds at 95°C, 30 seconds at 60°C and 30 seconds at 72°C. The cycle was followed
by a melt-curve. Standard curves were prepared by making a 1:5 serial dilution of
the input for each ChIP. ChIP and mock (rabbit serum) samples were assessed in triplicate
and the percentage of quantitative PCR product normalized and determined from the
standard curve using Bio-Rad CFX Manager 2.1 software (Biorad). Each ChIP experiment
and all PCR assessments were replicated on at least three independent occasions. Anti-Ctcf
ChIP on mouse TSCs derived from a C57BL/6J × CAST/EiJ cross was combined with next-generation
sequencing (100-bp paired-end reads) as described in detail elsewhere (Calabrese JM
and Magnuson T, in preparation). Briefly, ChIP was performed on 10 to 40 × 106 feeder-free TSCs. Cells were crosslinked for 10 minutes at room temperature in 0.6%
formaldehyde before quenching in 125mM glycine for 5 minutes. Cells were resuspended
in 50 mM Tris-HCl pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% Na-deoxycholate
and 0.1% SDS. Cells were sonicated to generate fragments averaging 200 to 500 bp,
cleared by centrifugation and resuspended at 20 × 106 cells/ml in the buffer above supplemented with 1% Triton-X100. ChIP was performed
with 10 μg of antibody. Post-ChIP, three washes with the buffer used for the ChIP
were performed, followed by a wash in the same buffer but with 500 mM NaCl, once with
20 mM Tris pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% Na-deoxycholate and once with TE buffer.
Chromatin was eluted for 15 minutes at 65°C in 50 mM Tris pH 8.0, 10 mM EDTA and 1%
SDS. A ChIP-Seq library was prepared according to Illumina instructions using 10 to
200 ng of ChIP DNA and sequenced on Illumina's Genome Analyzer IIx or HiSeq2000 instrument.
Ctcf ChIP-Seq data have been deposited with Gene Expression Omnibus and assigned the
provisional accession number GSE40667. The DNA sequence of the mouse Dxz4 array was
used to extract ChIP-Seq hits with homology to Dxz4. An approximately 232-bp DNA fragment
spanning the putative mouse Dxz4 Ctcf binding site was amplified from C57BL/6J and
castaneous genomic DNA isolated from tail snips. PCR was performed using HotStar Taq
(Qiagen) with an initial denaturation of 10 minutes at 94°C, followed by 35 cycles
of: 94°C for 30 seconds, 58°C for 30 seconds and 72°C for 30 seconds. The PCR product
was cloned into pDrive, and for each DNA source over 100 clones were isolated and
sequenced. Sequence variants specific to C57BL/6J and castaneous were then used to
manually align with 100% sequence identity over a minimum of 30 bp to the Ctcf ChIP-Seq
Dxz4 sequences and designated either C57BL/6J or castaneous. All SNP variants have
been deposited with dbSNP. Details can be found in Additional file 9.

Additional file 9.Dxz4 SNP data. List of SNPs identified in proximity to the Dxz4 Ctcf site in BL6 and cast DNA that
were used to assign Ctcf ChIP-Seq fragments to the BL6 or cast chromosome in Figure
5c.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

BPC conceived the study, analyzed and interpreted data, performed experiments, and
wrote the manuscript. AHH performed experiments and analyzed the data. MC performed
Ctcf ChIP-Seq and analyzed the data. CRM and DT carried out experiments. All authors
reviewed and contributed to the manuscript.

Acknowledgements

This work was supported by grants from the National Institute of General Medical Sciences
to BPC (NIH R01 GM073120) and TRM (NIH R01 GM10974). We are grateful to Danielle Maatouk
and Blanche Capel for assistance with derivation of mouse embryonic fibroblasts and
to Laura Carrel for use of the BC06 cell line. We are indebted to A Thistle for critically
evaluating the manuscript.